1. Field of the invention:
This invention relates to varieties of modified human PSTI and to DNA sequences encoding the same.
2. Description of the prior art:.
Two types of trypsin inhibitor are known which are derived from the pancreas, i.e., pancreatic secretory trypsin inhibitor (PSTI) and basic pancreatic trypsin inhibitor (BPTI). PSTI is present in all mammals, and is distributed not only in the pancreas but also in the kidney, lung, spleen, liver, brain and other organs. BPTI is distributed in various viscera of cows and other ruminants, but is not present in man or other mammals. Pubols et al. (J. Biol. Chem. 249, 2235, 1974) and Feinstein et al. (Eur. J. Biochem. 43, 569, 1974) have isolated and purified PSTI from human pancreatic juice, and Greene et al. (Methods Enzymol. 45. 813, 1976) determined the structure of this substance. Furthermore, Yamamoto et al. (Biochem. Biophys. Res. Commun. 132, 605, 1985) determined the DNA sequence corresponding to PSTI (FIG. 5). As shown by FIG. 5, human PSTI is a peptide composed of 56 amino acid residues, with a molecular weight of 6,242 daltons. It is known that sulfhydryl groups do not exist in PSTI, since the cysteine residues at positions 9 and 38, as well as 16 and 35, and also 24 and 56 are linked by disulfide bonds.
The trypsin inhibitor described above is present in the acinic cells of the pancreas, and in normal humans is secreted in the pancreatic juice together with various pancreatic enzymes so that it inhibits trypsin in the ductus pancreaticus. However, in acute pancreatitis, for some reason trypsin is activated and then trypsinogen and other enzyme precursors are activated in a chain reaction, and this presumably results in autodigestion of the pancreas. The administration of trypsin inhibitor is effective in the therapeutic treatment of this type of acute pancreatitis. The trypsin inhibitors currently used for this purpose include the above-mentioned bovine pancreatic BPTI as well as synthetic inhibitory agents, etc. In view of its source, human PSTI would appear to be the most appropriate trypsin inhibitor for use in this sort of therapy. However, since this form of PSTI has heretofore been prepared by isolation and purification from human pancreatic juice, sufficiently large quantities for therapeutic use could not be obtained, and therefore up to the present time human PSTI has not been employed in clinical practice. In order to solve this problem of quantitative production, the present inventors have developed a method of obtaining large quantities of human PSTI by applying recombinant DNA techniques (Japanese Laid-Open Patent Publication No. 62-253437). According to this method, human PSTI is expressed as a fusion protein with APH (aminoglycoside 3'-phosphotransferase II). This human PSTI fusion protein can be produced in large quantities in a microbial host, and after cleavage of this fusion protein with cyanogen bromide, human PSTI alone can be isolated and purified. The human PSTI obtained by this method possesses the same amino acid sequence as natural human PSTI, and therefore one may expect the same degree of therapeutic efficacy as that obtainable with natural PSTI in clinical applications. However, PSTI is also a peptide, and therefore with passage of time PSTI is gradually decomposed by proteolytic enzymes such as trypsin. Owing to this shortcoming, in order to achieve an adequately sustained trypsininhibiting effect, the quantity of PSTI which decomposes with passage of time must be monitored and replaced by an equal amount of the fresh substance, which has necessitated troublesome laboratory testing and other additional procedures.